Proteasome inhibitors like MG-132 sensitize cancer cells to ionizing radiation. In this study, we investigated interference of proteasome inhibition with NHEJ by caspase-3-dependent cleavage of DNA-PKcs as a possible underlying mechanism.
The relationship between pro-apoptotic pathways and radiosensitization is controversial, as is the importance of apoptosis in cell death following exposure to clinically relevant doses of ionizing radiation. In general, the majority of cells in solid carcinomas do not enter the apoptotic death pathway [
57]. Instead, they undergo several cell divisions until they die or finally survive. Thus, the fate of cells in solid carcinomas after irradiation may be determined more by their ability to repair DNA damage caused by ionizing radiation than by initiation of apoptosis [
58]. However, common molecular pathways may link these two phenomena. So far, any link between survival pathways and molecules involved in DNA repair has been elusive but cannot be excluded.
The most important DNA lesions occurring after exposure of cells to ionizing radiation that determines death or survival of a cancer cell are the double strand breaks. A process called non-homologous end joining (NHEJ) repairs these lesions in eukaryote cells. Concerted concession of NHEJ requires the activity of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), which is a known substrate of caspase-3. In this study we therefore focused on the possibility that proteasome inhibition by MG-132 activates caspase-3, as has been reported previously [
24,
30]. This could mediate apoptosis and cause degradation of DNA-PKcs, the catalytic subunit of DNA-PK [
20], resulting in radiosensitization. Reduction of DNA-PK activity following inactivation or mutation of DNA-PKcs is known to enhance radiosensitivity by decreasing repair of DNA-DSB's [
59‐
61]. Although DSB repair is critical for cell survival after exposure to ionizing radiation [
62], it is not clear whether this is a rate-limiting step dependent on the level of expression of DNA-PKcs. For example, DNA-PKcs level has been reported not to correlate with radiosensitivity of gliomas [
63] and normal fibroblasts [
64]. In any event, we were not able to detect meaningful changes in DNA-PK activity following MG-132 drug treatment. Degradation that did occur, appeared late at 24 hours and was probably an effect rather than a cause of the apoptotic process. According to the kinetics of the DSB-repair process described previously [
65], any event interfering with the repair of DNA-DSB's has to take place during the initial 6 hours after irradiation. Consistent with the late cleavage of DNA-PKcs, we were not able to detect early activation of caspase-3 following treatment of PC-3 cells with MG-132. In fact, we observed a substantial drop in DEVD-AMC cleavage activity, which might be explained by the observation, that proteasome activity is necessary to activate caspase-3 at least in some cells [
66,
67]. Our observations are in accordance with data from Hideshima and coworkers who also could not detect changes in DNA-PKcs levels or caspase-3 activation during the initial 6 hours of Valcade-treatment in multiple myeloma cells [
68]. Additionally, Wu and coworkers excluded the involvement of caspases in apoptosis of MO7e cells following proteasome inhibition, as caspase inhibitors failed to prevent DNA fragmentation [
20] and it is therefore possible that proteasome inhibition induces caspase-independent apoptosis as described for cells, defective in the ubiquitin pathway [
69].
Taken together, we conclude that although proteasome inhibition induces apoptosis in most cancer cells, sensitization of PC-3 cells to ionizing radiation occurs through mechanism that does not involve cleavage of DNA-PKcs.